PROJECT SUMMARY In this multi-PI proposal we will investigate the role of epigenetic mechanisms in driving severe asthma pathogenesis. The identification of molecular players/pathways that lead to pathogenic immune responses in asthma will be beneficial for the discovery of drug targets and development of new vaccine strategies to prevent asthma. Genome-wide enhancer profiling is a powerful new tool that has allowed investigators to precisely track genes and their cognate enhancers and thus capture molecular events involved in cellular development and differentiation. We have successfully micro-scaled this tool to enable large-scale human studies that have the potential to unravel novel molecular mechanisms underlying human diseases. In this proposal, we will define asthma-related epigenetic markers in ~100 patients with severe asthma, and correlate these with clinical phenotypes to obtain specific molecular markers for distinguishable disease states (i.e. severe asthma phenotypes). We will capitalize on three longitudinal asthma cohorts: (i) the Wessex AsThma CoHort of difficult asthma (WATCH), an observational clinical cohort with on-going active recruitment (~25 new cases/month) that currently consists of ~250 subjects with severe asthma, who are actively followed up by the treating physicians (PI Ramesh/Hasan, UK), (ii) The NIH-funded IOW, UK cohort of >100 well-phenotyped mild asthmatics; (iii) the La Jolla Institute (LJI) cohort of 40 subjects with mild-to-moderate asthma, who have provided blood samples every 6 months over two years. In Aim 1, we will profile the enhancer landscape in immune cell types relevant to severe asthma pathogenesis (nave, TH2 memory T cell subsets and classical monocytes), isolated from longitudinally collected blood samples from 200 subjects with varying asthma severity. We will perform comparative bioinformatics analysis to identify the immune cell enhancers linked to severe asthma. We will also perform transcriptional profiling of blood and airway immune cells to precisely define the genes that are affected (influenced) by the enhancers identified in severe asthma. In Aim 2, we will define the target genes of severe asthma associated enhancers and determine if genetics variants linked to asthma risk affect their function. We will then test the function of these genes in primary human T cells using optimized micro-scaled immunological assays. Overall, our discovery-based epigenetic studies and follow up functional studies will identify novel genes and pathways involved in severe asthma and corticosteroid insensitivity, and could open novel ways to treat severe asthma.